The long term goal of the PI's laboratory is to define the molecular components and mechanism of the store- operated Ca2+ entry (SOCE), which is known to be activated upon depletion of intracellular Ca2+ stores in excitable and non-excitable cells The focus of this innovative proposal is on calcium influx factor (CIF), a notoriously elusive and still un- identified messenger, that is produced upon depletion of the stores and was found in all living cells tested so far (from yeast to humans). We have unique knowledge and extensive experience in working with CIF extracts. We had discovered the physiological target for CIF (a specific plasma membrane bound Ca2+ independent phospholipase A2), and demonstrated its crucial role in SOCE, which was confirmed by other investigators. Most recently, we identified STIM1 as a trigger for CIF production in the endoplasmic reticulum. During our previous studies that focused on the mechanism of CIF production, action and its role in SOCE pathway, PI's laboratory had accumulated unique knowledge and expertise that may finally allow molecular identification of this extremely important but still elusive messenger. We had established new methods for advanced multi-step purification and biological testing of CIF, and can obtain sufficient quantities of CIF of the highest purity that is essential for its instrumental analysis. The goal of our new proposal is molecular identification of CIF. We understand that this is an ambitious goal, but we believe that we finally have everything for its successful achievement: 1) the knowledge about the molecular trigger of CIF production, and about physiological target of CIF, 2) extensive experience in working with CIF extracts, 3) new abundant source of CIF, 4) all the tools and novel approaches for its fine purification, 5) new effective and sensitive bioassay approaches and methods for testing samples and candidate molecules for CIF activity, 6) state-of-the-art facilities and established collaborations with the world class experts in mass spectrometry, NMR and SOCE mechanism. The feasibility of CIF purification and identification of its molecular structure is fully supported by our unique expertise, and extensive preliminary data. Specific Aim of this proposal is to identify CIF. We will: 1.1. Determine chemical and structural components of CIF using advanced mass spectrometry and NMR techniques. 1.2. Synthesize CIF molecule and its inactive analogs. 1.3. Verify CIF identity in bioassay systems, and match it with endogenous CIF activity. 1.4. Identify the biochemical pathway for CIF production. PUBLIC HEALTH RELEVANCE: The goal of this proposal is to identify calcium influx factor (CIF), a novel second messenger that is ubiquitously produced by all the cell types tested so far (from yeast to human), and is involved in activation of the store-operated Ca2+ entry pathway, one of the major mechanisms that determine Ca2+ homeostasis in health and disease. The feasibility of CIF identification is fully supported by our unique expertise, extensive preliminary data and advanced approaches that are already developed in PI's lab.